GSM7711791: K_F1_ABRR_rep3; Rutilus rutilus x Abramis brama; RNA-Seq
Fish were investigated for the presence of parasites one month after oncomiracidia infection (or following cohabitation) by fish dissection, and parasites were counted. Head kidney was collected from individual fish including 4-5 non-infected control specimens per each fish line and at least 4 P. homoion-infected specimens per each of five fish lines (R. rutilus, A. brama, F1 generation with A. brama mtDNA, backcross generation of F1 hybrid with A. brama mtDNA and paternal A. brama, backcross generation of F1 hybrid with A. brama mtDNA and paternal R. rutilus) in the case of OK experiment, six fish lines in the case of PH experiment including F1 generation with R. rutilus mtDNA. Total RNA was isolated from the head kidney of each fish specimen. For extraction, PureLink RNA Mini Kit (Ambion) with Trizol reagent (Thermo Fisher Scientific) and on-column PureLink DNase treatment were used according to the manufacturer´s protocol. Reagent and buffer volumes were adjusted according to the weight of tissue entering the isolation process (15.3 mg on average). The final elution was performed using 100 µl of RNAse-free water in the first step and the primal eluate in the second step. The yield and concentration of RNA isolates were checked using a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) and Qubit RNA HS Assay Kit (Thermo Fisher Scientific). The quality and integrity of RNA were analyzed using RNA 6000 Nano Kit on a 2100 Bioanalyzer instrument (Agilent Technologies). All RNA isolates were normalized by dilution at a uniform concentration of 20 ng/µl with RNase-free water. They served as templates for DNA library preparation and for the reverse transcription of total RNA into single-stranded cDNA using High Capacity RNA-to-cDNA Kit (Applied Biosystems by Thermo Fisher Scientific) in twice the reaction volume recommended by the manufacturer. All fish samples (RNA integrity number – RIN > 7) were used for DNA library preparation. 500 ng of total RNA was used for mRNA enrichment using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Subsequently, NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®, and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2, New England Biolabs) were used for library preparation, with 11 PCR cycles utilized for PCR enrichment. RNA fragmentation (13 minutes at 94°C) and the size selection conditions (a bead volume of 30 µl and 15 µl for the first and second bead selections, respectively) were further modified in the protocol. The quantification of DNA libraries was performed on a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) using Qubit dsDNA HS Assay Kit, and quality and size control were performed on a 2100 Bioanalyzer with DNA 1000 Kit (Agilent Technologies). Finally, amplicons were pooled in equimolar amounts. The final concentration of each particular library in the pool was 10nM in the pool. Libraries were sequenced by Macrogen Europe B.V. (Amsterdam, Netherlands) on an Illumina NovaSeq6000 system (one line on an S4 flow cell) in a paired-end configuration producing 150 bp long reads.